Molecular chaperones, including the Hsp70 and Hsp40 heat shock protein families, are required for numerous cellular processes. The long-term goal of the proposed research is to understand the role of these chaperones in the fundamental process of protein translation. Using the budding yeast Saccharomyces cerevisiae as a model system, the investigation will focus on an Hsp40 family member, Sis1, whose activity is essential for yeast cell viability and growth. A combination of genetic and biochemical techniques will be employed to analyze the function of Sis1 protein in protein synthesis. First, cell fractionation and protein analysis will be used to determine the distribution of Sis1 protein with respect to ribosomes and ribosome-associated factors. A genetic approach will be used to identify domains of Sis 1 that modulate its interactions with ribosomes and partner proteins. Second, the molecular basis for Sis1 function in translation will be investigated. An in vitro translation system will be developed, which will allow for direct analysis of Sis1-dependent translation initiation and will support biochemical manipulations such as protein immunodepletion and reconstitution. The proposed studies will provide information on fundamental translational control mechanisms, and uncover new physiological roles for molecular chaperones. The results of these studies will be directly applicable to human cell biology, since all eukaryotes share the same basic translation process and since the yeast Sis1 protein is functionally conserved in higher eukaryotes.